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To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Primary sensory cortical areas continuously receive thalamic inputs that arrive at different frequencies depending on the amount of sensory activity. The cortical response to repeated sensory stimuli rapidly adapts and different frequencies recruit cortical neuronal networks to different extents. GABAergic inhibition limits the spread of excitation within cortical neuronal networks. However, it is unknown how frequency adaptation of cortical network activity at different frequencies is shaped by GABAergic inhibition. Here, we find that in acute slices of visual cortex area V1 GABAergic inhibition affects frequency adaptation depending on the frequency of activity. Using voltage-sensitive dye imaging, we found that while increasing inhibitory postsynaptic currents (IPSCs) with flunitrazepam dampened the spread of cortical excitation, short-term adaptations to different stimulation frequencies were differentially affected. At high frequencies (40Hz), facilitation of cortical excitation was no longer transient, but facilitation was sustained. At low frequencies (10Hz) flunitrazepam decreased a depression of the excitation. In contrast, in mice lacking the GABAA receptor alpha1 subunit facilitation was reduced and depression enhanced. These findings suggest that GABAergic inhibition affects cortical excitation at different frequencies differentially, favoring facilitation at higher frequencies of excitation. [Copyright &y& Elsevier]

A robust increase in dopaminergic transmission in the nucleus accumbens (NAc) shell has been reported to be consistently associated with the long-term expression of behavioural sensitization to drugs of abuse. However, little is known about how this affects the neuronal network of the NAc. We made cellular recordings in NAc slices of saline- and amphetamine-pretreated adult rats and found that expression of behavioural sensitization was associated with long-lasting changes in the basal firing pattern of cholinergic interneurons up to 3 weeks after the last drug injection. Consequently, upon amphetamine sensitization, an inhibiting effect of the nicotinic receptor blocker mecamylamine on the amplitudes of spontaneous GABAergic synaptic currents as well as on the failure rate of electrically evoked GABAergic currents was found that was not present under control conditions. Thus, behavioural sensitization to amphetamine is associated with an up-regulation of the endogenous activation of nicotinic receptors that, in turn, stimulate the GABAergic synaptic transmission within the NAc shell. This is a new mechanism by which drugs of abuse may induce alterations in the processing and integration of NAc inputs involved in psychomotor sensitization. [ABSTRACT FROM AUTHOR]